Thrombospondin-1 (TSP-1) is an extracellular glycoprotein that plays a role in neoplasia, cell growth, and differentiation. We have examined the regulation of TSP-1 mRNA in cells expressing the v-src oncogene. Rat1 fibroblasts constitutively transformed by v-src expressed TSP-1 mRNA at levels that were 10- to 50-fold lower than those observed in parental, vector-transfected control cells. To analyze the kinetics of this effect, we used a line of BALB/c 3T3 fibroblasts containing a thermolabile v-src gene. Prolonged culture of these cells at the permissive temperature also resulted in down-regulation of TSP-1 mRNA. However, at early time points after temperature shift of growth-arrested cells, we observed a 3- to 15-fold increase in TSP-1 mRNA. This induction was abolished by the tyrosine kinase inhibitor, herbimycin-A, but not by the protein synthesis inhibitor, cycloheximide. The induction of TSP-1 by v-src occurred at a transcriptional level, as determined by nuclear run-on assays. Furthermore, the effect was mediated in part by a short region of the TSP-1 promoter which contains only 41 base pairs of 5' flanking DNA and 48 base pairs of the first exon. We conclude that, while overexpression of v-src results in brief transcriptional induction of TSP-1, the ultimate result of v-src transformation, at least in rodent fibroblasts, is repression of TSP-1 gene expression.